The limiting factor to using high-throughput sequencing to directly analyze de novo mutations over a short time span is the error rate of sequencing machines and PCR. Whereas eukaryotes mutate less than 1 in 109 bases per generation, the most accurate Illumina sequencing machines can misread 1 in 100 bases per generation. A number of recent methods have sought to lower this error rate. By introducing primers with random DNA “barcodes,” it is possible to read redundant copies of an original genomic molecule and form a consensus sequence robust to sequencing machine errors. However, because of stochasticity in PCR amplification, this method can be of low yield, i.e. an average of 30× coverage is required for a single consensus sequence. The accuracy of this method is also limited by the accuracy of polymerases used for sample preparation PCR. These errors can be overcome using duplex barcoding. Further, even when a small region is targeted, duplexing results in lower yield than traditional barcoding, requiring on average 1000× coverage per consensus sequence. The mutational landscape of an entire RNA virus was recently mapped using “circle sequencing,” but, the accuracy of this method is limited by the length of sequencing reads, which, for example, does not exceed 150 bp on an Illumina HiSeq.
Therefore, there exists a need for new techniques that may address the deficiencies in the existing methods for detection of genomic mutations.